We have been able to establish that the method of obtaining zero time H+/O ratios based on extrapolation of data obtained after 0.8 s back to zero gives undependable results. This is because although both [H+] vs t data, and [O] vs t data can be fit by single exponentials, neither is truly single exponential in character. This finding might help resolve some of the long standing controversy as to whether the zero-time value obtained by extrapolation procedures is 6.0 or 8.0 when succinate is the substrate. A final group of experiments and controls on the direct measurement of H+/O ratios accompanying cytochrome c oxidation at site III of the respiratory chain were analyzed by computer. These results substantiate earlier findings of a burst of H+/O ratios within the first 300 ms of a respiratory pulse. Preliminary experiments have shown that light scattering measurements can be dynamically recorded with a light pipe and photocell during a respiratory pulse, using mitochondria. These changes are related to mitochondrial size and energy state and will be the basis for a new system using rapid changes in external ion probe concentrations to determine Delta Psi and DeltapH during a pulse in mitochondrial respiration. Steps were taken to develop a system using caged H+ and O2 compounds to cause a step change in [H+] and [O2] in stirred solutions. This will enable the determination of electrode relaxation times in situ in the same stirred vessels used for respiratory pulse experiments.